We have shown that the first nonstructural protein in the DEN2 genome, NS1, can trans complement lethal mutations of this protein in the full-length "infectious" cDNA clone. We have continued studies on the role of NS1 in virus replication and spread. We originally generated a series of six deletion mutations in the carboxy teminus of the NS1 protein. Five of these mutations, JES32 - JES36, were sequential five amino acid deletions spanning 25 amino acids just upstream from the caroboxy terminus of the NS1 protein. The sixth deletion mutant, JES37, contained a deletion which removed all twenty five amino acids. The large 25 amino acid deletion mutant and four of the smaller deletion mutants were unable to grow in control LLC-MK2 cells. In contrast, JES36, was able to grow in control cells. All of the mutants were able to grow in LLC-MK2 cells expressing wildtype NS1. Analysis of NS1 dimer formation for these mutant viruses showed that only JES36 was able to form dimers. This suggests that the region being mutated was essential for NS1 dimer formation and that dimers are required for efficient replication of the virus. In addition to these mutations, one clone, JES36-2, was identified which contained the five amino acid deletion in NS1 and a single point mutation in NS2A. JES36-2 was able to grow in the NS1 complementing cell line, but the additional mutation in NS2A was lethal for growth of this mutant in control cells. A virus containing only the point mutation in NS2A, JES50, was able to replicate in both NS1 expressing cells and control cells. The NS2A mutation alone was not lethal for virus replication, whereas the additional mutation in NS1 created a virus which was only able to replicate in the NS1 complementing cells. In vitro studies on viral protein synthesis for the double mutant, JES36-2, showed that NS1 was properly processed and that NS1 from this virus was able to form dimers. These results suggest an additional requirement for NS2A in virus replication which may or may not be dependent on NS1. Further studies are underway to understand the role of NS2A in the proper processing of the NS1 protein from the dengue polyprotein, and the potential interaction of NS1 and NS2A in the virus replication machinery. We have also continued studies on the enhanced growth of wildtype dengue 2 virus in LLC-MK2 cells expressing dengue 2 NS1. Initial studies on NS1 expressing cells infected with dengue 2 showed that the NS1 expressing cells were resistant to virus induced apoptosis. Further studies on virus induced apoptosis in control and NS1 expressing cells show that NS1 expressing cells are resistant to apoptosis induced by actinomycin D and C2-ceramide. We have initiated biochemical studies to understand the interaction between NS1 and the cellular proteins which regulate apoptosis. In addition to biochemical studies on apoptosis, we are generating a series of point mutations in the NS1 gene of the full-length infectious dengue 2 cDNA clone. Viruses containing these mutations will be analyzed for their ability to replicate and spread in cells in culture. Levels of RNA replication and virus release will be studied. Changes in viral induced apotosis will also be analyzed in cells infected with these viruses.